1. Field of The Invention
Applicant's invention relates to the field of polychromatic polarimetric analysis of test specimens to determine the identity and concentration of the constituent elements or compounds of the test specimen. The invention relates more specifically to an apparatus and method for utilizing spectral transmission signatures involving polarization analysis for known compounds to identify and quantify those compounds in unknown test specimens.
2. Background Information
Presently, there are several different methods employed for identifying constituent compounds in a test specimen and determining the concentration of each compound. Chemical analysis of a specimen is frequently undertaken and usually yields excellent results. However, certain specimens, including internal bodily fluids, are not well suited for chemical analysis, because they can only be chemically analyzed by undertaking an invasive procedure that may be painful and may risk infection. For this reason, an accurate, non-invasive analytical method is needed for determining the identity and the concentration of various contituent compounds, such as glucose, alcohol or narcotic substances, in internal bodily fluids, such as blood.
Among the most promising tools for performing a non-invasive analytical procedure is by using spectrophotometric analysis. Spectrophotometric analysis relies on the principle that every compound has a unique "pattern" determined by the amount of light absorbed (or transmitted or reflected) by the compound at different wavelengths. Typically, analytical spectro-photometric methods target the specimen with light of known intensity, and measure the absorption of light by the specimen, at various wavelengths, or conversely, measure the intensity of light passing through the specimen, at various wavelengths, and then compare this "pattern" of absorption (or intensity) at different wavelengths with the known pattern of absorption per wavelength of various compounds.
Unfortunately, typical spectrophotometric analysis of a specimen is only of limited usefulness when the specimen is complex, (i.e. contains several compounds or elements), or if the density of the specimen is unknown, because absorption of light (or intensity of transmitted light) may be directly affected by these variable factors. Thus, relying solely on the absorption of light at various wavelengths does not yield a sufficiently accurate non-invasive method for analysis of bodily fluids. Certainly, the detection and measurement of other optical factors, which are unique for each compound, in addition to measurement of the absorption of light by the specimen per wavelength, would greatly improve the efficiency and accuracy of any spectroscopic technique used for determining the identity and concentration of constituent elements in a specimen.
In an attempt to provide a more accurate test and overcome other limitations of conventional spectrophotometric analysis of a specimen, several procedures have been developed using polarimetric analysis. Polarimetric analysis uses polarized light rather than partially polarized polychromatic light to irradiate the specimen and relies on the principle that specimens containing an optically active compound, such as glucose, will rotate the plane of polarized light, thereby causing a measurable "shift" in the plane of polarization. The degree and direction of the polarization shift that is caused by a compound is unique for each compound. In addition, certain compounds "depolarize" polarized light in is a unique manner.
Thus, by irradiating a test specimen with light that is polarized in a predetermined plane and then measuring the polarization shift and/or the degree of depolarization of tie light caused by the constituent components of the specimen at various wavelengths, the identity of the components in a specimen, as well as the respective concentrations of the components, theoretically can be more readily determined than by measuring only the absorption of light at each measured wavelength. Examples of prior-issued patents which are known to Applicant, and which relate to the use of polarized light in performing a spectroscopic analysis of a specimen, include the following:
U.S. Pat. No. 3,724,952 issued to Vossberg on Apr. 3, 1973, describes an apparatus and method for polarimetric analysis of a specimen, comprising the use of light that is polarized in one plane prior to passage of the light through the specimen. After the polarized light passes through the specimen, it passes through an analyzer and detector, which determine the "polarization shift" caused by the components of the specimen, as well as the degree of depolarization and the absorption of light by the specimen.
U.S. Pat. No. 4,901,728 and U.S. Pat. No. 5,009,230 issued to Hutchinson on Feb. 20, 1990 and Apr. 23, 1991, respectively, describe a device for non-invasive determination of blood glucose of a patient, by passing two orthogonal and equally polarized states of infrared light of the same intensity through a specimen and then passing the light through a polarizer to determine the rotation of the polarized light caused by the glucose in the specimen. The polarization shift is measured by calculating the difference in intensity of the two states of polarized light exiting the polarizer.
U.S. Pat. No. 4,011,014 issued to Tanton on Mar. 8, 1977 describes a machine for testing the rotation of polarized light by translucent specimens, that includes a polarizer to polarize light prior to the light contacting the specimen, and then measuring the polarization shift and other optical factors, that are caused by the specimen.
Each of the above-described methods and apparatuses for polarimetric analysis relies exclusively upon the principle of irradiating the specimen with light that is already polarized in a predetermined plane, and then measuring the polarization shift or other variables caused by the rotation of the polarized light by optically active compounds in the specimen. Although using polarized light to measure the "polarization" shift and/or other data dependent on the rotation of polarized light by the specimen, does provide certain information that can be objectively measured, in addition to the factors presented by standard spectrophotometric analysis, the use of light that already is polarized to irradiate the specimen has severe drawbacks.
For example, polarizing the light prior to irradiating the specimen significantly decreases the amount of light actually reaching the specimen, because a percentage of the light will be reflected or absorbed by the polarizing means before the light reaches the specimen. Obviously, when the specimen is dense, this loss of light could dramatically impact the amount of light actually passing through the specimen and capable of being measured.
In addition, polarizing the light in a particular plane of polarization before the light reaches the specimen, effectively eliminates all other planes of polarization in which the light travels, thereby drastically reducing the potential data that could be gathered if the light targeted on the specimen was randomly polarized light. In essence, trying to identify a compound in a complex specimen, by considering only the optical factors of light traveling through the specimen in a single plane of polarization is analogous to trying to formulate an accurate voter opinion poll by considering the opinion of only one or two persons. Obviously, the more voters that are considered, the more accurate will be the poll.
The inadequacy of the limited information obtained by using polarized light to irradiate a specimen is especially evident when the specimen contains two or more compounds, because the compounds may cause similar polarization shifts in the specific polarization plane in which the light is polarized, thereby making it very difficult to determine the identity and concentration of the different compounds in the specimen. The presence of more than one compound in the specimen may also "mask" the polarization shift that is actually caused by the targeted compound sought to be identified, because the presence of other compounds in the specimen may cause an enhancement or decrease in the polarization shift at the specific polarization plane in which the light is polarized. This masking effect on the polarization shift may cause either the identity or the concentration of the targeted compound to be incorrectly determined.
Measuring the polarization "shift" of light also requires that a polarizer be physically placed upstream from the specimen, to polarize the light in a specific plane of polarization prior to irradiating the specimen, and a separate polarizer/analyzer be physically placed downstream of the specimen through which light exiting the specimen is passed. This required use of two polarizers clearly causes the device to be more cumbersome and expensive than an invention that only requires the use of one polarizer.
Additionally, practicing certain inventions, such as the inventions disclosed in the Hutchinson patents, that use two beams of polarized light to measure the polarization shift and other factors related to rotation of the polarized light caused by optically active elements in the specimen, complicates matters considerably, because dual mechanisms are necessary to control the optical variables for each beam, such as the intensity of light and angle of polarization.
As briefly shown by the foregoing, both conventional spectrophotometric analysis and polarimetric analysis of a test specimen are severely hampered by the limited amount of data that can be obtained by merely measuring the absorption of light per wavelength by the specimen or by using polarized light to irradiate the specimen. Clearly, a method and apparatus is needed that would identify and accurately measure a wider range of optical factors than is possible by using standard spectrophotometric analysis or polarimetric analysis.
A starting point to the solution of the problem lies in the well developed electro-optical probe technologies currently in use in university, industrial, and government laboratories. The sensitivity of such probes may be increased enormously by taking advantage of the wavelength dependence of the polarized light. All wavelength components of polychromatic light are polarized, but not in the same way, and each must be examined separately. Each wavelength responds differently to a specific optically active medium.
After adding the analysis of wavelength it is advantageous to add the more complex analysis of the polarization rotational characteristics that result from the irradiation of many substances, especially organic. In general, organic molecules are structured in spiraled form and have a definite helicity or handedness. It is this helicity which gives a molecule its ability to rotate the polarization of the incident light. For example, dextrose (d-glucose) is, by convention, right-handed since, when viewed from the perspective of light emerging from the sample, the polarization axis has rotated in a clockwise direction. On the other hand, levulose (fruit sugar) is left-handed since it rotates the polarization axis in a counter clockwise direction. Molecules or material which exhibit this kind of optical activity are said to possess optical rotary power. In particular, these are termed dextrorotary or levorotatory respectively depending upon the action on the polarization of the incident light. The magnitude of the angle, through which the polarization direction rotates is, in simple theory, proportional to the inverse of the wavelength of the incident light squared. Sometimes called a dispersion function, this relationship has a weak dependence on wavelength but is strongly a function of the type of material or molecular structure being irradiated. This functional dependence on the physical properties of the sodium manifests itself in the difference of the indices of refraction for right- and left-handed polarized light. Two circularly polarized waves of opposite helicity form a set of basic fields for the description of any general state of polarization. As a result, for example, if the polarization of the light irradiating the sample were purely elliptical not only would the ellipse rotate by about an axis parallel to the direction of propagation of the light, but the ellipse also distorts--its eccentricity changes. This latter phenomenon is called circular dichroism. It is due to the different absorption between right- and left-handed circularly polarized light.
In a fluid, where there is no long-range order, the molecules are randomly oriented. Nevertheless, the effect of rotary power is not averaged out to zero. Since the constituent molecules all have a definite helicity which is the same, they cannot be brought into coincidence with their mirror images--they are enantiomorhpous. Thus, the effect of the rotary power of an individual molecule is enhanced in a fluid state. Substances which exhibit both optical rotary power and circular dichroism are referred to as chiral media.
A glucose solution is an isotropic chiral substance. When plane-polarized light impinges normally on glucose the vibration ellipse of the transmitted light is different from the vibration ellipse of the incident light. The difference is characterized by two quantities: (i) Optical rotation (OR), which is the angle by which the transmission ellipse rotates with respect to the incidence ellipse; (ii) Circular dichroism (CD), which is a measure of the difference in the eccentricities of the two ellipses. Profiles of the OR and the CD of an isotropic chiral substance with respect to frequency are sufficiently unique that they can be used as a component in the signature of a substance to be identified. Because the OR and the CD of any substance have been shown to be Kramers-Kronig-consistent, complete knowledge of either of the two quantities as a function of the frequency is sufficient to determine the other; therefore, the more easily measured OR is often used to characterize isotropic chiral substances.
A first issue that must be addressed is that of polarization of the light incident on the biological sample whose glucose content has to be monitored. Let us suppose that the incident light is a planewave traveling in the +z direction (of a cartesian coordinate system) at a frequency f. The electric field phasor associated with this planewave may be adequately set up as EQU E.sub.inc (z,t)=A.sub.x u.sub.x +A.sub.y u.sub.y !e.sup.-i2.pi.f(t-z/.spsp.c.sbsp.0.sup.), (1)
where t is time and c.sub.0 =3.times.10.sup.8 m/s is the speed of light in free space; i=.sqroot.(-1); (u.sub.x, u.sub.y, u.sub.z) are the unit cartesian vectors; and A.sub.x and A.sub.y are complex amplitudes with units of V/m.
Let the complex amplitudes be independent of time t. In general, Eq. (1) then represents an elliptically polarized planewave whose vibration ellipse does not change with time t. When either A.sub.x =0 or A.sub.y =0, the planewave is said to be linearly polarized. When A.sub.x =.+-.iA.sub.y, the planewave is circularly polarized.
Suppose now that A.sub.x and A.sub.y are functions of time t. Then Eq. (1) should be rewritten as EQU E.sub.inc (z,t)=A.sub.x (t)u.sub.x +A.sub.y (t)u.sub.y !e.sup.-i2.pi.f(t-z/.spsp.c.sbsp.0.sup.). (2)
It still denotes a planewave, but one whose vibration ellipse changes with time t. Complicated sources have to be utilized in order to deliver specific A.sub.x (t) and A.sub.y (t). Indeed, the prior art devices utilize a complicated light source that yields A.sub.x (t) and A.sub.y (t) as controllable functions of time t.
The preferred embodiment of the present invention, however, utilizes a source based on Quartz-Tungsten-Halogen (QTH) lamp whose output in the focal region is partially polarized. Other suitable light sources include devices which emit light at multiple frequencies, such as LEDs. To understand the term "partially polarized", it is best to begin by thinking about "totally unpolarized" planewaves. The functions A.sub.x (t) and A.sub.y (t) are continuously random functions of time for a totally unpolarized planewave, therefore the rotation of a totally unpolarized planewave by a glucose cell cannot be measured and even the concept is of no meaning.
A partially polarized planewave can be thought of as a combination of a totally unpolarized planewave and an elliptically polarized planewave. The second component of the partially polarized wave suffers a definite rotation on passage through a glucose cell, therefore can be used for OR measurements.
The present invention has a source that delivers a slightly polarized planewave, thus its rotation by the glucose cell is meaningful.
A second issue that must be addressed is that of chromaticity. The devices described in the prior art ideally need monochromatic sources, i.e., sources whose outputs are fixed at precisely one frequency. Practical monochromatic sources cannot be ideal, instead their frequency range is very small.
Suppose f.sub.c is the center-frequency of a source and its 3-db bandwidth in denoted by .DELTA.f; then, we can define a quality factor EQU Q=f.sub.c /.DELTA.f. (3)
The QTH lamp used in the preferred eabodiment of the present invention is a white-light lamp operating from 400 to 2000 nm with a peak at 900 nm; thus, its useful frequency spectrum ranges from 1.5.times.10.sup.14 Hz to 7.5.times.10.sup.14 Hz with its peak intensity at 3.3.times.10.sup.14 Hz. As the QTH output is roughly independent of the frequency over the operating range, we can estimate its Q=3.3/(7.5-1.5)=0.55. Thus, the QTH lamp is definitely a polychromatic source.
The present invention also utilizes a polarization-preserving analyzer whose response is flat over the 2.3.times.10.sup.14 Hz to 4.3.times.10.sup.14 Hz range, and it uses a compensated polychromatic detector to measure the intensity of the beam transmitted by the analyzer. In sum, the present invention is polychromatic (low-Q), while the devices described in the prior art are monochromatic (high-Q).
Polychromaticity has a definite advantage over monochromaticity for such things as blood glucose measurements. The OR spectrum of a chiral solute in a non-chiral solvent depends on the concentration of the solute. The amount of glucose in a (diabetic) biological sample varies with time. A polychromatic system therefore has a much better chance of monitoring a continuously varying non-normoglycemic sample than a monochromatic one.